Kinetics of halide release of haloalkane dehalogenase: Evidence for a slow conformational change

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides, The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity, but the k(cat) values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s(-1), respectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall k(cat). Furthermore, at 5 mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s(-1) (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5 mM 1,2-dibromoethane occurred at a rate of only 8 s(-1). The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of k(obs) on the halide concentration suggested that there were two parallel routes for halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations. The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s(-1) for bromide and chloride, respectively). We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion. A solvent kinetic isotope effect of (H2O)-H-2 was found both on k(cat) and on the rate of halide release. (H2O)-H-2 mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of (H2O)-H-2 on the conformation of proteins.